Excessive T3 in the early postnatal days can induce hearing loss in mice
To evaluate the effect of T3 treatments at different postnatal periods on auditory function in mice, ABR was performed at P18. Compared with the control group, the ABR-click thresholds increased significantly in P0 or P1 group, while T3 given at P3 (P3 group) had no significant effects on ABR thresholds (Fig. 1A). Mice in P0 group showed severe deafness with mean thresholds above 80 dB SPL at 8–40 kHz, while mice in P1 group displayed moderate to severe deafness with hearing thresholds at 8, 16, 24, 32 and 40 kHz of 61.3 ± 6.3, 51.3 ± 2.5, 57.5 ± 9.6, 73.8 ± 7.5, 90.0 ± 0dB SPL, respectively (Fig. 1B). Mice treated with T3 at P3 showed normal hearing at P18 (Fig. 1B).
Excessive T3 accelerate the maturation of the greater epithelial ridge (GER) and do not affect hair cell survival
HC loss is a major cause of hearing loss. Thus, we analyzed the survival patterns of HCs in T3-treated mice. No substantial HC loss was observed in different T3 treated group at P18 (Fig. 2A–L). Although scattered losses of outer HCs were occasionally observed in the basal turn of P0 group (white arrows, Fig. 2F), statistical analysis showed that the number of OHCs was not significantly changed compared to the control group (n = 4, P > 0.05) (Fig. 2M).
In neonatal mice, the cochlea proceeded to develop structurally and functionally before hearing onset, and regression of the GER is a prominent structural changing event. During natural development, cells in GER promote the development and maturation of sensory epithelium through programmed cell death. We performed activated casapase-3 staining to determine the apoptosis pattern of GER in T3-treated mice. At P6, no activated caspase-3-positive (caspase-3+) cells were detected in the GER of control mice, while a large number of caspase-3+ cells were captured in GER of the T3-treated group (Fig. 2O–R). In contrast, caspase-3+ cells were evident in the GER of control cochleae and were not detected in the T3-treated group at P11 (Fig. 2S–V). Statistical analysis showed that the number of caspase-3+ cells differed significantly between the two groups at P6 and P11 (n = 4, P < 0.01) (Fig. 2N).
Excessive T3 interferes with the morphology of HC’s stereocilia and function of MET channel
In mammals, stereocilia are located in the cuticular plate of the cochlear sensory cells and are responsible for converting mechanical vibrations generated by sound stimulation into electrical signals. Structural or functional defects of the stereocilia are one of the main causes of congenital or progressive deafness. We performed SEM to characterize the morphology of stereocilia in all turns of the cuticular plate in T3-treated mice. In the control group, three rows of stereocilia formed a V-shaped bundle in all three turns (Fig. 3A–C, a–c). However, treatment with T3 at P0 or P1 resulted in the stereocilia bundle of outer HCs in the apical and middle turns being disordered and losing their V-shaped structure, although there were no obvious changes in morphology of the stereocilia bundle in the basal turn (Fig. 3D–I, d–i). In contrast the morphology and arrangement of the HC stereocilia bundle was almost unaffected when T3 was given at P3 (Fig. 3J–L, j–l). These results suggest that the abnormal arrangement of the HC stereocilia bundle may be strongly associated with hearing loss caused by excessive T3. In addition, FM1-43 loading of HCs was used to assess functional of MET channel. Compared with the control group, the uptake of FM1-43 by OHCs in the T3 treatment group was reduced (Fig. 3M–P). Quantitative results showed that the relative fluorescence density of FM1-43 in OHCs of T3-treated mice decreased by 23.9 ± 13.9% (Fig. 3Q). These results indicated that abnormalities of the HC stereocilia bundles and dysfunctions of MET channel might be responsible for the hearing loss induced by excessive T3.
Excessive T3 can induced overproduction of Deiter-like cells
To investigate the effect of T3 on remodeling of the OC in mice. Mice in P0 group were sacrificed at P18 and the SCs were labeled with Sox2. Furthermore, phalloidin was used to label the feet of the DCs and PCs. In the control group, the DCs were neatly arranged in three rows and the PCs were arranged in a single row in all turns (Fig. 4A–F). However, in P0 group, we observed four rows of DCs in the apical and middle turns, indicating the production of excess Deiter-like cells. In addition, the arrangement of Sox2-labeled SCs was disordered and the OPCs were jagged compared to the control group (Fig. 4G–J). The arrangement of DCs in the basal turn was almost unaffected in P0 group (Fig. 4K, L). Statistical analysis showed that the number of DCs was significantly increased in the apical and middle turns (n = 4, P < 0.001) (Fig. 4M). We also quantified the distance between inner pillar cells (IPCs) and OPCs and found that the relative distance between the feet of the IPCs and OPCs was reduced in the apical and middle turns P0 group, but there was no significant difference in the basal turn (Fig. 4N). Furthermore, we labeled DCs with Cx30, a protein subunit that constitutes gap junctions, which serves as a functional marker of DCs. In the control group, Cx30 signals (green) were evenly distributed along the boundaries of all DCs (Fig. 5B, D, F), whereas, in the T3 treatment group, we observed that the extra Deiter-like cells also expressed Cx30, which suggested that these cells might have partial functions of DCs (Fig. 5H, J, h, j). We quantified the number of Cx30+ DCs in all three turns of both groups, and found that the number of Cx30+ cells in the apical and middle turns of the T3 treatment group was significantly increased (n = 4, P < 0.001) (Fig. 5M).
Next, we explored the effects of excessive T3 administration at other time-points after birth on development of the OC in mice. When T3 was given at P1, we again observed the four rows of Cx30-expressing DCs in the apical and middle turns (Fig. 6D, d, E, e). However, T3 given at P3 did not significant effect the number of DCs (Fig. 6G–I, g–i). Quantitative results showed that the number of DCs was significantly increased in apical and middle turns from the P1 group (n = 4, P < 0.001) (Fig. 6J). The distance between the feet of the IPCs and OPCs was also reduced in the apical and middle turns of the P1 group (Fig. 6K). These parameters did not change significantly in the P3 group. Our results reveal that excessiveT3 can regulate the development of OC, especially for DCs, in a narrow postnatal time window.
Ultrastructural changes of SCs in T3 treated mice
Axial sections of the cochlea revealed the nuclei of three rows of DCs in the control group (Fig. 7A, B). However, in the apical turn of the T3-treated group, we clearly observed nuclei of four rows of DCs (Fig. 7C, D). Acetylated α-tubulin was used to label DCs and PCs, and this a proper maker to measure the supporting capacity of DC and PC. Three-dimensional reconstructed images showed that more than three rows of phalangeal processes were observed emanating from the foot of DCs in the T3-treated group (Fig. 7E, F). In addition, ultrastructural examination showed the presence of three rows of DC cell bodies in the control group and bundles of microtubules and normal mitochondria in the DCs (Fig. 7G–I). In P0 group, we observed four rows of DC cell bodies (Fig. 7J). The phalangeal processes of the extra DCs (DC4) showed normal architecture of the bundles of microtubules and mitochondria (Fig. 7K, L), which indicated that the overproduced Deiter-like cell have the similar structure and function as normal DCs.
Characterization of gene expression changes in the cochleae of T3-treated mice by realtime qPCR
In order to investigate the mechanism of T3 involved in the remodeling of the OC, we performed qPCR to analyze the expression levels of a series of genes regulating the development of inner ear. The mRNA expression of Atoh1 and Sox2, two transcription factors that regulate HCs and SCs’ development, was significantly up-regulated (Fig. 8A). However, the other important factors Pou4f3, Neurog1, and Gfi1 did not change significantly. In addition, we analyzed the Notch, Wnt, TGFβ and FGF signaling pathways as well as cell cycle signaling pathways and found that the transcription levels of Notch pathway-related genes, such as Notch1, Notch2, Notch2, Notch3, Jag1, Jag2, Hey1, Hey2, Hes1, Hes5 and Dll1 were significantly down-regulated (Fig. 8B). The expression of FGF and most TGFβ signaling pathway genes did not change significantly, while only Smad4, Bmpr1b and Ltbp1 were downregulated (Fig. 8C, D). In the Wnt pathway, the mRNA expression levels of Lgr5 and Wnt2b were significantly down-regulated and other related genes were not significantly changed (Fig. 8E). In addition, we found that the cell cycle-dependent kinases Cdk2 and Cdk4, and cell division cyclin Cdc25c, were down-regulated in cochleae of T3-treated mice (Fig. 8F). All these results suggest that T3 may lead to overproduction of DCs mainly through down-regulation of the Notch signaling pathway in early cochlear development.
Downregulation of the Notch signaling pathway did not aggravate the overproduction of DCs induced by T3
Based on the above PCR results, we speculated that the Notch signaling pathway might be responsible for the overproduction of DCs induced by T3. To verify whether T3 combined with DAPT, an inhibitor of the Notch pathway, to synergistically regulate the additional increase of DCs, we treated mice with T3 and DAPT in combination, and then evaluated the DCs in the different groups at P18 (Fig. 9A). In the control and DAPT alone treated groups, three rows of DCs were neatly arranged, and Cx30 was observed at the edge of all DCs (Fig. 9B–G, b–g), which suggested that DAPT treatment alone did not affect the number of DCs. In the T3 and T3 combined with DAPT (T3+DAPT) treatment groups, four rows of DCs were observed in the apical and middle turns (Fig. 9H, I, K, L). Quantitative results showed no significant difference in the number of DCs between the groups treated with T3 or T3+DAPT (Fig. 9N). In addition, we measured the distance between the feet of the IPCs and OPCs and found that the distance did not significantly differ between the groups treated with T3 and with T3+DAPT (Fig. 9M). These results suggest that T3 combined with inhibition of Notch signaling did not aggravate the overproduction of DCs induced by T3.
Effects of excessive T3 combined with down-regulated Sox2 on the remodeling of organ of Corti
Recent studies have shown that Sox2-CreER mice exhibit Sox2 haploid deficiency due to one of the alleles being replaced by CreER. Using this characteristic, Sox2CreER/+ mice were injected with T3 to explore the effect of T3 combined with Sox2 downregulation on the development of SCs in the inner ear (Fig. 10A). In Sox2CreER/+ mice, three rows of DCs were neatly arranged, and Cx30 was observed at the edge of all DCs, almost as in the control group (Fig. 10B–G, b–g). In the T3 and the Sox2CreER/+ + T3 groups, four rows of DCs were observed in the apical and middle turns, and the quantified results showed no significant difference in the number of DCs between the T3 and the Sox2CreER/+ + T3 groups (Fig. 10N). However, two rows of OPCs were observed in the apical and middle turns of the Sox2CreER/+ + T3 group (Fig. 10K, k, L, l). Statistical analysis showed that the number of OPCs was significantly increased in the apical and middle turns of the Sox2CreER/+ + T3 group (n = 4, P < 0.01) (Fig. 10O). These results suggest that T3 combined with Sox2 downregulation did not aggravate the overproduction of DCs induced by T3, but did induce overproduction of OPCs. In addition, additional OPCs appear to form new TC (Fig. S1F).